1. Technical Field
The present invention relates to a method for producing an L-amino acid by fermentation using a microorganism. Specifically, the present invention relates to a method for producing L-amino acids such as L-lysine, L-arginine, L-ornithine, L-histidine, L-isoleucine, L-threonine, L-proline, L-phenylalanine, L-cysteine, and L-glutamic acid. These are industrially useful L-amino acids. Namely, L-lysine, L-threonine, L-isoleucine, and L-proline are useful as additives for animal feed, components of health food, and amino acid infusions. L-arginine and L-ornithine are useful as liver function-promoting agents, amino acid infusions, and components of comprehensive amino acid preparations. L-histidine is useful as a liver function-promoting agent and as a precursor of histamine. L-phenylalanine is useful as a precursor of sweeteners.
2. Background Art
L-amino acids are industrially produced by fermentation using microorganisms belonging to the genus Brevibacterium, Corynebacterium, Escherichia, or the like.
Methods for producing L-lysine have been reported in EP 0857784A, JP 11-192088A, WO00/53726, and WO96/17930. Methods for producing L-arginine have been reported in EP 0999267A, EP 1170358A, and JP 2002-017342A. In these reported methods, basic L-amino acid-producing bacteria strains were used, including strains separated from nature or artificially mutated strains thereof, and recombinant strains which have enhanced activity of a basic L-amino acid biosynthetic enzyme.
Furthermore, methods for producing L-amino acids from methanol, which is available for fermentation in large amounts for low cost, using a mutated or genetically-modified microorganism strain belonging to the genus Methylophilus or Methylobacillus have also been reported (WO00/61723 and JP 2001-120269A).
Methods of modifying uptake or export of L-amino acids in bacterial cells have been known to improve the L-amino acid-producing ability of the bacteria. Methods of modifying L-amino acid uptake include eliminating or decreasing uptake of an L-amino acid into cells to enhance L-amino acid-producing ability. Specifically, these methods include a method of deleting the gluABCD operon, or a part thereof, to eliminate or attenuate uptake of L-glutamic acid (EP 1038970A).
Methods of modifying exporter include eliminating or reducing export of an intermediate or a substrate of L-amino acid biosynthesis, and a method of enhancing export of a produced L-amino acid. As a method of eliminating or reducing export of an intermediate of L-glutamic acid biosynthesis, a method of mutating or disrupting α-ketoglutarate permease gene to reduce a export of α-ketoglutaric acid is known (WO01/005959).
As a method of enhancing an L-amino acid export, a method of enhancing lysE (a gene for basic L-amino acid exporter; J. Mol. Microbiol. Biotechnol., 1999 November; 1(2):327-36) in a strain of Corynebacterium bacterium is known for producing L-lysine (WO97/23597) or L-arginine (US Patent Publication 2003-0113899). A method of enhancing the expression of rhtA, B, C gene (JP 2000-189177A) and yfiK, yahN gene (EP 1016710A), which have been suggested to be involved in export of L-amino acids, in cells of Escherichia bacterium are also known.
As a gene for export of basic L-amino acids, the aforementioned lysE gene is known. However, when a lysE gene is amplified in a methanol-assimilating bacterium such as Methylophilus bacterium, and the resulting strain is used for production of L-lysine or L-arginine, a wild-type lysE gene derived from a Coryneform bacterium is lethal for the Methylophilus bacterium, and thus it is necessary to introduce a mutant lysE gene (EP 1266966A) that allows growth of the host microorganism. Therefore, the lysE gene cannot always function in export of L-lysine or L-arginine when it is introduced into heterogeneous microorganisms. Therefore, it is desirable to obtain a gene for L-amino acid exporter and production that exhibits an ability to export sufficient amounts of L-amino acids, including L-lysine and L-arginine, in a variety of heterogeneous host microorganisms.
The ybjE gene is located on the genome of Escherichia coli and has been predicted to encode a putative surface protein (Science, 277 (5331):1453-74, 1997). However, cloning of the gene and analysis thereof through expression in bacterial cells has not been reported, and thus its physiological function has remained unknown.